Method for treating sensorineural hearing loss using glial cell line-derived neurotrophic factor (GDNF) protein product

ABSTRACT

The present invention relates generally to methods for preventing and/or treating injury or degeneration of cochlear hair cells and spiral ganglion neurons by administering glial cell line-derived neurotrophic factor (GDNF). The invention relates more specifically to methods for treating sensorineural hearing loss.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods for preventing and/ortreating injury or degeneration of inner ear sensory cells, such as haircells and auditory neurons, by administering glial cell line-derivedneurotrophic factor (GDNF) protein product. The invention relatesspecifically to methods for preventing and/or treating hearing loss dueto variety of causes.

Neurotrophic factors are natural proteins, found in the nervous systemor in non-nerve tissues innervated by the nervous system, that functionto promote the survival and maintain the phenotypic differentiation ofcertain nerve and/or glial cell populations (Varon et al., Ann. Rev.Neuroscience, 1:327, 1979; Thoenen et al., Science, 229:238, 1985).Because of this physiological role, neurotrophic factors are useful intreating the degeneration of such nerve cells and the loss ofdifferentiated function that results from nerve damage. Nerve damage iscaused by conditions that compromise the survival and/or proper functionof one or more types of nerve cells, including: (1) physical injury,which causes the degeneration of the axonal processes (which in turncauses nerve cell death) and/or nerve cell bodies near the site ofinjury, (2) temporary or permanent cessation of blood flow (ischemia) toparts of the nervous system, as in stroke, (3) intentional or accidentalexposure to neurotoxins, such as the cancer and AIDS chemotherapeuticagents cisplatinum and dideoxycytidine, respectively, (4) chronicmetabolic diseases, such as diabetes or renal dysfunction, or (5)neurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease, and Amyotrophic Lateral Sclerosis, which result from thedegeneration of specific neuronal populations. In order for a particularneurotrophic factor to be potentially useful in treating nerve damage,the class or classes of damaged nerve cells must be responsive to thefactor. It has been established that all neuron populations are notresponsive to or equally affected by all neurotrophic factors.

The first neurotrophic factor to be identified was nerve growth factor(NGF). NGF is the first member of a defined family of trophic factors,called the neurotrophins, that currently includes brain-derivedneurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6(Thoenen, Trends. Neurosci., 14:165-170, 1991; Snider, Cell, 77:627-638,1994; Bothwell, Ann. Rev. Neurosci., 18:223-253, 1995). Theseneurotrophins are known to act via the family of trk tyrosine kinasereceptors, i.e., trkA, trkB, trkC, and the low affinity p75 receptor(Snider, Cell, 77:627 -638, 1994; Bothwell, Ann. Rev. Neurosci.,18:223-253, 1995; Chao et al., TINS 18:321-326, 1995).

Glial cell line-derived neurotrophic factor (GDNF) is a recentlydiscovered protein identified and purified using assays based upon itsefficacy in promoting the survival and stimulating the transmitterphenotype of mesencephalic dopaminergic neurons in vitro (Lin et al.,Science, 260:1130-1132, 1993). GDNF is a glycosylated disulfide-bondedhomodimer that has some structural homology to the transforming growthfactor-beta (TGF-β) super family of proteins (Lin et al., Science,260:1130-1132, 1993; Krieglstein et al., EMBO J., 14:736-742, 1995;Poulsen et al., Neuron, 13:1245-1252, 1994). GDNF mRNA has been detectedin muscle and Schwann cells in the peripheral nervous system (Hendersonet al., Science, 266:1062-1064, 1994; Trupp et al., J. Cell Biol.,130:137-148, 1995) and in type I astrocytes in the central nervoussystem (Schaar et al., Exp. Neurol., 124:368-371, 1993). In vivo,treatment with exogenous GDNF stimulates the dopaminergic phenotype ofsubstantia nigra neurons and restores functional deficits induced byaxotomy or dopaminergic neurotoxins in animal models of Parkinson'sdisease (Hudson et al., Brain Res. Bull., 36:425-432, 1995; Beck et al.,Nature, 373:339-341, 1995; Tomac et al., Nature, 373:335-339, 1995;Hoffer et al., Neurosci. Lett., 182:107-111, 1994). Although originallythought to be relatively specific for dopaminergic neurons, at least invitro, evidence is beginning to emerge indicating that GDNF may have alarger spectrum of neurotrophic targets besides mesencephalicdopaminergic and somatic motor neurons (Yan and Matheson, Nature373:341-344, 1995; Oppenheim et al., Nature, 373:344-346, 1995; Mathesonet al., Soc. Neurosci. Abstr, 21, 544, 1995; Trupp et al., J. CellBiol., 130:137-148, 1995). In particular, GDNF was found to haveneurotrophic efficacy on brainstem and spinal cord cholinergic motorneurons, both in vivo and in vitro (Oppenheim et al., Nature,373:344-346, 1995; Zurn et al., Neuroreport, 6:113-118, 1994; Yan etal., Nature, 373: 341-344, 1995; Henderson et al., Science,266:1062-1064, 1994), on retinal neurons, such as photoreceptors invitro (currently pending U.S. application Ser. No. 08/564,833 by Louis,filed Nov. 29, 1995) and retinal ganglion cells both in vitro and invivo (currently pending U.S. application Ser. No. 08/564,458 by Yan,filed Nov. 29, 1995 and both in vitro and in vivo on sensory neuronsfrom the dorsal root ganglion both (currently pending U.S. applicationSer. No. 08/564,844 (by Yan et al.) filed Nov. 29, 1995).

Of general interest to the present invention is WO93/06116 (Lin et al.,Syntex-Synergen Neuroscience Joint Venture), published Apr. 1, 1993,which reports that GDNF is useful for the treatment of nerve injury,including injury associated with Parkinson's disease. Also of interestare a report in Schmidt-Kastner et al., Mol. Brain Res., 26:325-330,1994 that GDNF mRNA became detectable and was upregulated afterpilocarpine-induced seizures; reports in Schaar et al., Exp. Neurol.,124:368-371, 1993 and Schaar et al., Exp. Neurol., 130:387-393, 1994that basal forebrain astrocytes expressed moderate levels of GDNF mRNAunder culture conditions, but that GDNF did not alter basal forebrainChAT activity; and a report in currently pending U.S. application Ser.No. 08/535,682 filed Sep. 28, 1995 that GDNF is useful for treatinginjury or degeneration of basal forebrain cholinergic neurons. GDNF hasnot previously been shown to promote survival, regeneration orprotection against degeneration of inner ear cells such as hair cellsand auditory neurons.

The neuroepithelial hair cells in the organ of Corti of the inner ear,transduce sound into neural activity, which is transmitted along thecochlear division of the eighth cranial nerve. This nerve consists offibers from three types of neurons (Spoendlin, H. H. In: Friedmann, I.Ballantyne, J., eds. Ultrastructural Atlas of the Inner Ear; London,Butterworth, pp. 133-164, 1984): 1) afferent neurons, which lie in thespiral ganglion and connect the cochlea to the brainstem. 2) efferentolivocochlear neurons, which originate in the superior olivary complexand 3) autonomic adrenergic neurons, which originate in the cervicalsympathetic trunk and innervate the cochlea. In the human, there areapproximately 30,000 afferent cochlear neurons, with myelinated axons,each consisting of about 50 lamellae, and 4-6 μm in diameter. Thishistologic structure forms the basis of uniform conduction velocity,which is an important functional feature. Throughout the length of theauditory nerve, there is a trophic arrangement of afferent fibers, with`basal` fibers wrapped over the centrally placed `apical` fibers in atwisted rope-like fashion. Spoendlin (Spoendlin, H. H. In: Naunton, R.F., Fernadex, C. eds. Evoked Electrical Activity in the Auditory NervousSystem. London, Academic Press, pp. 21-39, 1978) identified two types ofafferent neurons in the spiral ganglion on the basis of morphologicdifferences: type I cells (95%) are bipolar and have myelinated cellbodies and axons that project to the inner hair cells. Type II cells(5%) are monopolar with unmyelinated axons and project to the outer haircells of the organ of Corti. Each inner hair cell is innervated by about20 fibers, each of which synapses on only one cell. In contrast, eachouter hair cell is innervated by approximately six fibers, and eachfiber branches to supply approximately 10 cells. Within the cochlea, thefibers divide into: 1) an inner spiral group, which arises primarilyipsilaterally and synapses with the afferent neurons to the inner haircells, and 2) a more numerous outer radial group, which arises mainlycontralaterally and synapses directly with outer hair cells. There is aminimal threshold at one frequency, the characteristic or bestfrequency, but the threshold rises sharply for frequencies above andbelow this level (Pickles, J. O. In: Introduction to the Physiology ofHearing. London, Academic Press, pp. 71-106, 1982). Single auditorynerve fibers therefore appear to behave as band-pass filters. Thebasilar membrane vibrates preferentially to different frequencies, atdifferent distances along its length, and the frequency selectivity ofeach cochlear nerve fiber is similar to that of the inner hair cell towhich the fiber is connected. Thus, each cochlear nerve fiber exhibits aturning curve covering a different range of frequencies from itsneighboring fiber (Evans, E. F. In: Beagley H. A. ed. Auditoryinvestigation: The Scientific and Technological basis. New York, OxfordUniversity Press, 1979). By this mechanism, complex sounds are brokendown into component frequencies (frequency resolution) by the filters ofthe inner ear.

Hearing loss of a degree sufficient to interfere with social andjob-related communications is among the most common chronic neuralimpairments in the US population. On the basis of health-interview data(Vital and health statistics. Series 10. No. 176. Washington, D.C. (DHHSpublication no. (PHS) 90-1504), it is estimated that approximately 4percent of people under 45 years of age and about 29 percent of those 65years or over have a handicapping loss of hearing. It has been estimatedthat more than 28 million Americans have hearing impairment and that asmany as 2 million of this group are profoundly deaf (A report of thetask force on the National Strategic plan. Bethesda, Md.: NationalInstitute of Health, 1989). The prevalence of hearing loss increasesdramatically with age. Approximately 1 per 1000 infants has a hearingloss sufficiently severe to prevent the unaided development of spokenlanguage (Gentile, A. et al. Characteristics of persons with impairedhearing: United States, 1962-1963. Series 10. No. 35. Washington, D.C.:Government printing office, 1967 (DHHS publication no. (PHS) 1000)(Human communication and its disorders: an overview. Bethesda, Md.:National Institutes of health, 1970). More than 360 per 1000 personsover the age of 75 have a handicapping hearing loss (Vital and healthstatistics. Series 10. No. 176. Washington, D.C. (DHHS publication no.(PHS) 90-1504).

It has been estimated that the cost of lost productivity, specialeducation, and medical treatment may exceed $30 billion per year fordisorders of hearing, speech and language (1990 annual report of theNational Deafness and other Communication Disorders Advisory Board.Washington, D.C.: Government Printing Office, 1991. (DHHS publicationno. (NIH) 91-3189). The major common causes of profound deafness inchildhood are genetic disorders and meningitis, constitutingapproximately 13 percent and 9 percent of the total, respectively(Hotchkiss, D. Demographic aspects of hearing impairment: questions andanswers. 2nd ed. Washington, D.C.: Gallaudet University Press, 1989). Inapproximately 50 percent of the cases of childhood deafness, the causeis unknown, but is likely due to genetic causes or predisposition(NanceW E, Sweeney A. Otolaryngol. Clin. North Am 1975; 8: 19-48).

Impairment anywhere along the auditory pathway, from the externalauditory canal to the central nervous system, may result in hearingloss. The auditory apparatus can be subdivided into the external andmiddle ear, inner ear and auditory nerve and central auditory pathways.Auditory information in humans is transduced from a mechanical signal toa neurally conducted electrical impulse by the action of approximately15,000 neuroepithelial cells (hair cells) and 30,000 first-order neurons(spiral ganglion cells) in the inner ear. All central fibers of spiralganglion neurons form synapses in the cochlear nucleus of the pontinebrainstem. The number of neurons involved in hearing increasesdramatically from the cochlea to the auditory brain stem and theauditory cortex. All auditory information is transduced by only 15,000hair cells, of which the so-called inner hair cells, numbering 3500, arecritically important, since they form synapses with approximately 90percent of the 30,000 primary auditory neurons. Thus, damage to arelatively few cells in the auditory periphery can lead to substantialhearing loss. Hence, most causes of sensorineural loss can be ascribedto lesions in the inner ear (Nadol, J. B., New England Journal ofMedicine, 1993, 329: 1092-1102).

Hearing loss can be on the level of conductivity, sensorineural andcentral level. Conductive hearing loss is caused by lesions involvingthe external or middle ear, resulting in the destruction of the normalpathway of airborne sound amplified by the tympanic membrane and theossicles to the inner ear fluids. Sensorineural hearing loss is causedby lesions of the cochlea or the auditory division of the eight cranialnerve. Central hearing loss is due to lesions of the central auditorypathways. These consist of the cochlear and dorsal olivary nucleuscomplex, inferior colliculi, medial geniculate bodies, auditory cortexin the temporal lobes and interconnecting afferent and efferent fibertracts (Adams R. D. and Maurice, V. Eds. in: Principles of Neurology.1989. McGraw-Hill Information services Company. PP 226-246).

As mentioned previously, at least 50 percent of cases of profounddeafness in childhood have genetic causes (Brown, K. S. Med. Clin. NorthAM. 1969; 53: 741-72). If one takes into consideration the probabilitythat genetic predisposition is a major causative factor inpresbycusis--or age-related hearing loss--which affects one third of thepopulation over 75 years of age (Nadol, J. B. In: Beasley D S, Davis GA, eds. Aging: Communication Processes and Disorders. New York: Grune &Stratton, 1981:63-85), genetic and hereditary factors are probably thesingle most common cause of hearing loss. Genetic anomalies are muchmore commonly expressed as sensorineural hearing loss than as conductivehearing loss. Genetically determined sensorineural hearing loss isclearly a major, if not the main cause of sensorineural loss,particularly in children (Nance W E, Sweeney A. Otolaryngol. Clin. NorthAm 1975; 8: 19-48). Among the most common syndromal forms ofsensorineural loss are Waardenburg's syndrome, Alport's syndrome andUsher's syndrome.

A variety of commonly used drugs have ototoxic properties. The bestknown are the aminoglycoside antibiotics (Lerner, S. A. et al eds.Aminoglycoside ototoxicity. Boston: Little, Brown, 1981; Smith, C. R. etal. N Engl. J. Med. 1980; 302: 1106-9), loop diuretics (Bosher, S. K.,Acta Otolaryngol. (Stockh) 1980; 90: 4-54), salicylates (Myers, E. N. atal. N Engl. J. Med. 1965; 273:587-90) and antineoplastic agents such ascisplatin (Strauss, M. at al. Laryngoscope 1983; 143:1263.5).Ototoxicity has also been described during oral or parenteraladministration of erythromycin (Kroboth, P. D. at al. Arch. Intern Med.1983; 1:169-79; Achweitzer, V. G., Olson, N. Arch. Otolaryngol. 1984;110:258-60).

Most ototoxic substances cause hearing loss by damaging the cochlea,particularly the auditory hair cells and the stria vascularis, aspecialized epithelial organ within the inner ear, that is responsiblefor the homeostasis of fluids and electrolytes (Nadol, J. B. New EnglandJ. Med. 1993, 329: 1092-1102). Secondary neural degeneration may occurmany years after an ototoxic event affecting the hair cells. There isevidence that some ototoxic substances may be selectively concentratedwithin the inner ear, resulting in progressive sensorineural lossdespite the discontinuation of systemic administration (Federspil, P. atal. J. Infect. Dis. 1976; 134 Suppl: S200-S205)

Trauma due to acoustic overstimulation is another leading cause ofdeafness. There is individual susceptibility to trauma from noise.Clinically important sensorineural hearing loss may occur in some peopleexposed to high-intensity noise, even below levels approved by theOccupational Safety and Health Agency (Osguthorpe, J. D. ed. WashingtonD.C.: American Academy of Otolaryngology-Head and Neck SurgeryFoundation, 1988).

Demyelinating processes, such as multiple sclerosis, may causesensorineural hearing loss (Noffsinger, D at al. Acta Otolaryngol Suppl(Stockh) 1972; 303:1-63). More recently, a form of immune-mediatedsensorineural hearing loss has been recognized (McCabe, B. F. Ann OtolRhinol Laryngol 1979; 88:585-9). The hearing loss is usually bilateral,is rapidly progressive (measured in weeks and months), and may or maynot be associated with vestibular symptoms.

A variety of tumors, both primary and metastatic, can produce either aconductive hearing loss, or a sensorineural hearing loss, by invadingthe inner ear or auditory nerve (Houck, J. R. et al. Otolaryngol HeadNeck Surg 1992; 106:92-7). A variety of degenerative disorders ofunknown cause can produce sensorineural hearing loss. Meniere's syndrome(Nadol, J. B. ed. Meniere's disease: pathogenesis, pathophysiology,diagnosis, and treatment. Amsterdam: Kugler & Ghedini 1989),characterized by fluctuating sensorineural hearing loss, episodicvertigo, and tinnitus, appears to be caused by a disorder of fluidhomeostasis within the inner ear, although the pathogenesis remainsunknown. Sudden idiopathic sensorineural hearing loss (Wilson, W. R. atal. Arch Otolaryngol 1980; 106:772-6), causing moderate-to-severesensorineural deafness, may be due to various causes, including innerear ischemia and viral labyrinthitis.

Presbycusis, the hearing loss associated with aging, affects more thanone third of persons over the age of 75 years. The most commonhistopathological correlate of presbycusis is the loss ofneuroepithelial (hair) cells, neurons, and the stria vascularis of theperipheral auditory system (Schuknecht H. F. Pathology of the Ear.Cambridge, Mass: Harvard University Press, 1974:388-403). Presbycusis isbest understood as resulting from the cumulative effects of severalnoxious influences during life, including noise trauma, ototoxicity andgenetically influenced degeneration.

Certain neurotrophic factors have been shown to regulate neuronaldifferentiation and survival during development (Korsching S. J.Neurosci. 13:2739-2748, 1993) and to protect neurons from injury andtoxins in adult (Hefti, Neurosci. 6:2155-2162, 1986; Apfel et al., AnnNeurol 29:87-89, 1991; Hyman et al., Nature 350:230-233, 1991; Knusel etal., J. Neurosci. 12:4391-4402, 1992; Yan et al., Nature, 360:753-755,1992; Koliatsos et al., Neuron, 10:359-367, 1993). In situ hybridizationstudies indicate that mRNAs for the neurotrophin receptors TrkB and TrkCare expressed by developing cochleovestibular ganglia (Ylikoski et al.,Hear. Res. 65:69-78 1993; Schecterson et al., Hearing Res. 73: 92-1001994) and that mRNAs for BDNF and NT-3 are found in the inner ear,including the organ of Corti (Pirvola et al., Proc. Natl. Acad. Sci. USA89: 9915-9919, 1992; Schecterson et al., Hearing Res. 73: 92-100 1994;Wheeler et al., Hearing Res. 73: 46-56, 1994). The physiological role ofBDNF and NT-3 in the development of the vestibular and auditory systemswas investigated in mice that carry a deleted BDNF and/or NT-3 gene(Ernfors et al., Neuron 14: 1153-1164 1995). In the cochlea, BDNFmutants lost type-2 spiral neurons, causing an absence of outer haircell innervation. NT-3 mutants showed a paucity of afferents and lost 87percent of spiral neurons, presumably corresponding to type-1 neurons,which innervate inner hair cells.

Double mutants had an additive loss, lacking all vestibular and spiralneurons. The requirement of TrkB and TrkC receptors for the survival ofspecific neuronal populations and the maintenance of target innervationin the peripheral sensory system of the inner ear was demonstrated bystudying mice carrying a germline mutation in the tyrosine kinasecatalytic domain of these genes (Schimmang et al., Development, 121:3381-3391 1995). Gao et al., (J. Neurosci. 15: 5079-5087, 1995) showedsurvival-promoting potency of NT-4/5, BDNF and NT-3 for rat postnatalspiral ganglion neurons in dissociated cultures and that NT-4/5protected these neurons from neurotoxic effects of the anti-cancer drug,cisplatin. Also, BDNF and NT-3 have been shown to support the survivalof adult rat auditory neurons in dissociated cultures (Lefebvre et al.,NeuroReport 5: 865-868, 1994).

There have been no previous reports of the use of GDNF in the treatmentof hearing loss. Since hearing impairment is a serious affliction, theidentification of any agent and treatment method that can protect theauditory neurons and hair calls from damage would be of great benefit.

SUMMARY OF THE INVENTION

The present invention provides methods for treating sensorineuralhearing loss comprising administering to a subject having a lesion inthe inner ear a therapeutically effective amount of a glial cellline-derived neurotrophic factor (GDNF) protein product. For example,the hearing loss may be associated with injury or degeneration ofneuroepithelial hair cells (cochlear hair cells) or spiral ganglionneurons in the inner ear.

The present invention is based on the discoveries that hair cellsrespond to GDNF by resisting the toxic effects of ototoxins, such ascisplatin and neomycin, and that auditory neurons also respond to GDNFby resisting the toxic effects of variety of ototoxins, such as forexample cisplatin, neomycin, and sodium salicylate. Thus, atherapeutically effective amount GDNF protein product may beadministered to promote the protection, survival or regeneration of haircells and spiral ganglion neurons.

It has also been discovered that lesions or disturbances to thevestibular apparatus may also be treated by administering to a subjecthaving such a lesion or disturbance a therapeutically effective amountof a GDNF protein product. Such lesions may result in dizziness, vertigoor loss of balance.

It is contemplated that such GDNF protein products would include a GDNFprotein such as that depicted by the amino acid sequence set forth inFIG. 1 (SEQ ID NO:1), as well as variants and derivatives thereof. It isalso contemplated that such GDNF protein products would include Met⁻¹!GDNF.

According to the invention, the GDNF protein product may be administeredparenterally at a dose ranging from about 1 μg/kg/day to about 100mg/kg/day, typically at a dose of about 0.1 mg/kg/day to about 25mg/kg/day, and usually at a dose of about 5 mg/kg/day to about 20mg/kg/day. It is also contemplated that, depending on the individualpatient's needs and route of administration, the GDNF protein productmay be given at a lower frequency such as weekly or several times perweek, rather than daily. It is further contemplated that GDNF proteinproduct may be administered directly into the middle ear or the innerear. One skilled in the art will appreciate that with suchadministration of a smaller amount of GDNF protein product may be used,for example, a direct middle ear or inner-ear administration dose in therange of about 1 μg/ear to about 1 mg/ear in a single injection or inmultiple injections.

It is further contemplated that GDNF protein product be administered incombination or conjunction with an effective amount of a secondtherapeutic agents, such as BDNF and NT-3. The invention also providesfor the use of GDNF protein product in the manufacture of a medicamentor pharmaceutical composition for the treatment of injury ordegeneration of hair cells and auditory neurons for the variety ofcauses of sensorineural hearing loss. Such pharmaceutical compositionsinclude topical, oral or middle and inner ear GDNF protein productformulations or in combination with cochlear implants.

It will also be appreciated by those skilled in the art that theadministration process can be accomplished via cell therapy and genetherapy means, as further described below. For example, in a genetherapy means cells have been modified to produce and secrete the GDNFprotein product. The cells may be modified vivo or in vivo. Numerousadditional aspects and advantages of the invention will become apparentto those skilled in the art upon consideration of the following detaileddescription of the invention which describes presently preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for preventing and/or treatingsensorineural hearing loss by administering a therapeutically effectiveamount of glial cell line-derived neurotrophic factor (GDNF) proteinproduct. According to one aspect of the invention, methods are providedfor treating damaged hair cells and auditory neurons by administering atherapeutically effective amount of GDNF protein product by means of apharmaceutical composition, the implantation of GDNF-expressing cells,or GDNF gene therapy. The invention may be practiced using anybiologically active GDNF protein product, including a GDNF proteinrepresented by the amino acid sequence set forth in SEQ ID NO:1,including variants and derivatives thereof. In addition to oral,parenteral or topical delivery of the GDNF protein product,administration via cell therapy and gene therapy procedures iscontemplated.

The present invention is based on the initial discoveries that GDNFprotects hair cells from ototoxins-induced cell death in explantcultures of rat's cochlea and dissociated spiral ganglion neurons fromadult rat in culture. It is contemplated that administration ofexogenous GDNF protein product will protect hair cells and spiralganglion neurons from traumatic damage (such as noise trauma and acuteor chronic treatments of cisplatin and aminoglycoside antibiotics) orfrom damage resulting from a lack of neurotrophic factors caused byinterruption of transport of the factors from the axon to the cell body.Such treatment is expected to allow hair cells and/or auditory neuronsto tolerate intermittent insults from either environmental noise trauma,treatments with ototoxins and to slow down the progressive degenerationof the auditory neurons and hair cells, that is responsible for hearingloss in pathological conditions such as presbycusis (age-related hearingloss), inherited sensorineural degeneration, and post-idiopathic hearinglosses and to preserve the functional integrity of the inner ear. Itwill also support the auditory neurons for a better and longerperformance of cochlear implants.

According to the invention, the GDNF protein product may be administeredinto the middle ear at a dose ranging from about 1 μg/kg/day to about100 mg/kg/day, typically at a dose of about 0.1 mg/kg/day to about 25mg/kg/day, and usually at a dose of about 5 mg/kg/day to about 20mg/kg/day. GDNF protein product may be administered directly into theinner ear in cases where invasion of the inner ear is already in placesuch as in the procedure of cochlear implant or surgeries of the innerear. In such cases, a smaller amount of GDNF protein product will beadministered, for example, from about 1 μg/ear to about 1 mg/ear in asingle injection or in multiple injections. In cases where a chronicadministration of the factor is needed, an Alzet mini-pump will beplaced attached to a cannula the tip of which will be introduced intothe middle or inner ear for a continuous release of the factor. GDNF canbe also developed in a form of ear-drops which will penetrate thetympanic membrane of the Bulla. It is further contemplated that GDNFprotein product be administered with an effective amount of a secondtherapeutic agent for the treatment of auditory neuron degeneration,together with BDNF and NT-3 as well as other factors or drugs usedcurrently or in the future for the treatment of the various inner earpathologies. A variety of pharmaceutical formulations and differentdelivery techniques are described in further detail below.

As used herein, the term "GDNF protein product" includes purifiednatural, synthetic or recombinant glial cell line-derived neurotrophicfactor, biologically active GDNF variants (including insertion,substitution and deletion variants), and chemically modified derivativesthereof. Also included are GDNFs that are substantially homologous tothe human GDNF having the amino acid sequence set forth in SEQ ID NO:1.GDNF protein products may exist as homodimers or heterodimers in theirbiologically active form.

The term "biologically active" as used herein means that the GDNFprotein product demonstrates similar neurotrophic properties, but notnecessarily all of the same properties, and not necessarily to the samedegree, as the GDNF having the amino acid sequence set forth in SEQ IDNO: 1. The selection of the particular neurotrophic properties ofinterest depends upon the use for which the GDNF protein product isbeing administered.

The term "substantially homologous" as used herein means having a degreeof homology to the GDNF having the amino acid sequence set forth in SEQID NO:1 that is preferably in excess of 70%, most preferably in excessof 80%, and even more preferably in excess of 90% or 95%. For example,the degree of homology between the rat and the human protein is about93%, and it is contemplated that preferred mammalian GDNF will have asimilarly high degree of homology. The percentage of homology asdescribed herein is calculated as the percentage of amino acid residuesfound in the smaller of the two sequences which align with identicalamino acid residues in the sequence being compared, when four gaps in alength of 100 amino acids may be introduced to assist in that alignment(as set forth by Dayhoff, in Atlas of Protein Sequence and Structure,Vol. 5, p. 124, National Biochemical Research Foundation, Washington,D.C. (1972), the disclosure of which is hereby incorporated byreference). In particular, Dayhoff describes that " i!n practice, tworelated proteins may be aligned with the insertion of an average of 3 or4 gaps in a length of 100 residues. About 20% of the aligned amino acidsare identical. Under these conditions, the statistical conclusion ofcommon ancestry can be drawn with great confidence. Common ancestry mayexist even though it cannot be proved from the comparison of twosequences. The use of additional evidence, such as the correspondence ofthe active sites, the comparisons of many related sequences with one newone, and the nature of the three-dimensional structures, will eventuallypermit the inference of relationships of even more remotely relatedstructures." Also included as substantially homologous is any GDNFprotein product which may be isolated by virtue of cross-reactivity withantibodies to the GDNF of SEQ ID NO:1 or whose genes may be isolatedthrough hybridization with the gene or with segments of the geneencoding the GDNF of SEQ ID NO:1.

The GDNF protein products according to this invention may be isolated orgenerated by any means known to those skilled in the art. Exemplarymethods for producing GDNF protein products useful in the presentinvention are described in U.S. application Ser. No. 08/182,183 filedMay 23, 1994 and its parent applications; PCT Application No.PCT/US92/07888 filed Sep. 17, 1992, published as WO 93/06116 (Lin etal., Syntex-Synergen Neuroscience Joint Venture); European PatentApplication No. 92921022.7, published as EP 610 254; and co-owned,co-pending U.S. application Ser. No. 08/535,681 filed Sep. 28, 1995("Truncated Glial Cell-Line Derived Neurotrophic Factor"), thedisclosures of which are hereby incorporated by reference.

Naturally-occurring GDNF protein products may be isolated from mammalianneuronal cell preparations, or from a mammalian cell line secreting orexpressing GDNF. For example, W093/06116 describes the isolation of GDNFfrom serum-free growth conditioned medium of B49 glioblastoma cells.GDNF protein products may also be chemically synthesized by any meansknown to those skilled in the art. GDNF protein products are preferablyproduced via recombinant techniques because they are capable ofachieving comparatively higher amounts of protein at greater purity.Recombinant GDNF protein product forms include glycosylated andnon-glycosylated forms of the protein, and protein expressed inbacterial, mammalian or insect cell systems.

In general, recombinant techniques involve isolating the genesresponsible for coding GDNF, cloning the gene in suitable vectors andcell types, modifying the gene if necessary to encode a desired variant,and expressing the gene in order to produce the GDNF protein product.Alternatively, a nucleotide sequence encoding the desired GDNF proteinproduct may be chemically synthesized. It is contemplated that GDNFprotein product may be expressed using nucleotide sequences which differin codon usage due to the degeneracies of the genetic code or allelicvariations. WO93/06116 describes the isolation and sequencing of a cDNAclone of the rat GDNF gene, and the isolation, sequencing and expressionof a genomic DNA clone of the human GDNF gene. WO93/06116 also describesvectors, host cells, and culture growth conditions for the expression ofGDNF protein product. Additional vectors suitable for the expression ofGDNF protein product in E. coli are disclosed in published EuropeanPatent Application No. EP 0 423 980 ("Stem Cell Factor") published Apr.24, 1991, the disclosure of which is hereby incorporated by reference.The DNA sequence of the gene coding for mature human GDNF and the aminoacid sequence of the GDNF is shown in FIG. 19 (SEQ ID NO:5) ofWO93/06116. FIG. 19 does not show the entire coding sequence for thepre-pro portion of GDNF, but the first 50 amino acids of human pre-proGDNF are shown in FIG. 22 (SEQ ID NO:8) of WO93/06116.

Naturally-occurring GDNF is a disulfide-bonded dimer in its biologicallyactive form. The material isolated after expression in a bacterialsystem is essentially biologically inactive, and exists as a monomer.Refolding is necessary to produce the biologically activedisulfide-bonded dimer. Processes for the refolding and naturation ofthe GDNF expressed in bacterial systems are described in WO93/06116.Standard in vitro assays for the determination of GDNF activity aredescribed in WO93/06116 and in co-owned, co-pending U.S. applicationSer. No. 08/535,681 filed Sep. 28, 1995, and are hereby incorporated byreference.

A. GDNF variants

The term "GDNF variants" as used herein includes polypeptides in whichamino acids have been deleted from ("deletion variants"), inserted into("addition variants"), or substituted for ("substitution variants"),residues within the amino acid sequence of naturally-occurring GDNF.Such variants are prepared by introducing appropriate nucleotide changesinto the DNA encoding the polypeptide or by in vitro chemical synthesisof the desired polypeptide. It will be appreciated by those skilled inthe art that many combinations of deletions, insertions, andsubstitutions can be made provided that the final molecule possessesGDNF biological activity.

Mutagenesis techniques for the replacement, insertion or deletion of oneor more selected amino acid residues are well known to one skilled inthe art (e.g., U.S. Pat. No. 4,518,584, the disclosure of which ishereby incorporated by reference.) There are two principal variables inthe construction of variants: the location of the mutation site and thenature of the mutation. In designing GDNF variants, the selection of themutation site and nature of the mutation will depend on the GDNFcharacteristic(s) to be modified. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconservative amino acid choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target amino acidresidue, or (3) inserting amino acid residues adjacent to the locatedsite. Conservative changes in from 1 to 20 amino acids are preferred.Once the amino acid sequence of the desired GDNF protein product isdetermined, the nucleic acid sequence to be used in the expression ofthe protein is readily determined. N-terminal and C-terminal deletionvariants may also be generated by proteolytic enzymes.

For GDNF deletion variants, deletions generally range from about 1 to 30residues, more usually from about 1 to 10 residues, and typically fromabout 1 to 5 contiguous residues. N-terminal, C-terminal and internalintrasequence deletions are contemplated. Deletions may be introducedinto regions of low homology with other TGF-β super family members tomodify the activity of GDNF. Deletions in areas of substantial homologywith other TGF-β super family sequences will be more likely to modifythe GDNF biological activity more significantly. The number ofconsecutive deletions will be selected so as to preserve the tertiarystructure of the GDNF protein product in the affected domain, e.g.,cysteine crosslinking. Non-limiting examples of deletion variantsinclude truncated GDNF protein products lacking from one to fortyN-terminal amino acids of GDNF, or variants lacking the C-terminalresidue of GDNF, or combinations thereof, as described in co-owned,co-pending U.S. application Ser. No. 08/535,681 filed Sep. 28, 1995,which is hereby incorporated by reference.

For GDNF addition variants, amino acid sequence additions typicallyinclude N-and/or C-terminal fusions ranging in length from one residueto polypeptides containing a hundred or more residues, as well asinternal intrasequence additions of single or multiple amino acidresidues. Internal additions may range generally from about 1 to 10residues, more typically from about 1 to 5 residues, and usually fromabout 1 to 3 amino acid residues. Examples of N-terminal additionvariants include GDNF with an N-terminal methionyl residue (an artifactof the direct expression of GDNF in bacterial recombinant cell culture),which is designated Met⁻¹ !GDNF, and fusion of a heterologous N-terminalsignal sequence to the N-terminus of GDNF to facilitate the secretion ofmature GDNF from recombinant host cells. Such signal sequences generallywill be obtained from, and thus be homologous to, the intended host cellspecies. Additions may also include amino acid sequences derived fromthe sequence of other neurotrophic factors. A preferred GDNF proteinproduct for use according to the present invention is the recombinanthuman Met⁻¹ !GDNF.

GDNF substitution variants have at least one amino acid residue of theGDNF amino acid sequence removed and a different residue inserted in itsplace. Such substitution variants include allelic variants, which arecharacterized by naturally-occurring nucleotide sequence changes in thespecies population that may or may not result in an amino acid change.Examples of substitution variants (see, e.g., SEQ ID NO: 50) aredisclosed in co-owned, co-pending U.S. application Ser. No. 08/535,681filed Sep. 28, 1995, and are hereby incorporated by reference.

Specific mutations of the GDNF amino acid sequence may involvemodifications to a glycosylation site (e.g., serine, threonine, orasparagine). The absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at anyasparagine-linked glycosylation recognition site or at any site of themolecule that is modified by addition of an O-linked carbohydrate. Anasparagine-linked glycosylation recognition site comprises a tripeptidesequence which is specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thror Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) result innon-glycosylation at the modified tripeptide sequence. Thus, theexpression of appropriate altered nucleotide sequences produces variantswhich are not glycosylated at that site. Alternatively, the GDNF aminoacid sequence may be modified to add glycosylation sites.

One method for identifying GDNF amino acid residues or regions formutagenesis is called "alanine scanning mutagenesis" as described byCunningham and Wells (Science, 244:1081-1085, 1989). In this method, anamino acid residue or group of target residues are identified (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Those domainsdemonstrating functional sensitivity to the substitutions then arerefined by introducing additional or alternate residues at the sites ofsubstitution. Thus, the target site for introducing an amino acidsequence variation is determined, alanine scanning or random mutagenesisis conducted on the corresponding target codon or region of the DNAsequence, and the expressed GDNF variants are screened for the optimalcombination of desired activity and degree of activity.

The sites of greatest interest for substitutional mutagenesis includesites where the amino acids found in GDNF proteins from various speciesare substantially different in terms of side-chain bulk, charge, and/orhydrophobicity. Other sites of interest are those in which particularresidues of GDNF-like proteins, obtained from various species, areidentical. Such positions are generally important for the biologicalactivity of a protein. Initially, these sites are substituted in arelatively conservative manner. Such conservative substitutions areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes (exemplary substitutions) are introduced, and/orother additions or deletions may be made, and the resulting productsscreened for activity.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Substitutions                                                      Original Preferred       Exemplary                                            Residue  Substitutions   Substitutions                                        ______________________________________                                        Ala (A)  Val             Val; Leu; Ile                                        Arg (R)  Lys             Lys; Gln; Asn                                        Asn (N)  Gln             Gln; His; Lys; Arg                                   Asp (D)  Glu             Glu                                                  Cys (C)  Ser             Ser                                                  Gln (Q)  Asn             Asn                                                  Glu (E)  Asp             Asp                                                  Gly (G)  Pro             Pro                                                  His (H)  Arg             Asn; Gln; Lys; Arg                                   Ile (I)  Leu             Leu; Val; Met; Ala;                                                           Phe; norleucine                                      Leu (L)  Ile             norleucine; Ile; Val;                                                         Met; Ala; Phe                                        Lys (K)  Arg             Arg; Gln; Asn                                        Met (M)  Leu             Leu; Phe; Ile                                        Phe (F)  Leu             Leu; Val; Ile; Ala                                   Pro (P)  Gly             Gly                                                  Ser (S)  Thr             Thr                                                  Thr (T)  Ser             Ser                                                  Trp (W)  Tyr             Tyr                                                  Tyr (Y)  Phe             Trp; Phe; Thr; Ser                                   Val (V)  Leu             Ile; Leu; Met; Phe;                                                           Ala; norleucine                                      ______________________________________                                    

Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleic acid sequences) areexpected to produce GDNF protein products having functional and chemicalcharacteristics similar to those of natural GDNF. In contrast,substantial modifications in the functional and/or chemicalcharacteristics of GDNF protein products may be accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Naturally occurring residues are dividedinto groups based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for another. Such substituted residues may beintroduced into regions of the GDNF protein that are homologous withother TGF-β super family proteins, or into the non-homologous regions ofthe molecule.

B. GDNF Derivatives

Chemically modified derivatives of GDNF or GDNF variants may be preparedby one of skill in the art given the disclosures herein. The chemicalmoieties most suitable for derivatization include water solublepolymers. A water soluble polymer is desirable because the protein towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. Preferably, the polymer will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically, and if so, thedesired dosage, circulation time, resistance to proteolysis, and otherconsiderations. The effectiveness of the derivatization may beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or, more preferably, by injection or infusion, or,further formulated for oral, pulmonary or other delivery routes), anddetermining its effectiveness.

Suitable water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone) polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weightranges from about 2 kDa to about 100 kDa for ease in handling andmanufacturing (the term "about" indicating that in preparations ofpolyethylene glycol, some molecules will weigh more, some less, than thestated molecular weight). Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofpolyethylene glycol on a therapeutic protein or variant).

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono-, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art. See for example, EP 0 401384, the disclosure of which is hereby incorporated by reference(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.,20:1028-1035, 1992 (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residue. Those having a free carboxyl group may includeaspartic acid residues, glutamic acid residues, and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecule(s). For therapeutic purposes,attachment at an amino group, such as attachment at the N-terminus orlysine group is preferred. Attachment at residues important for receptorbinding should be avoided if receptor binding is desired.

One may specifically desire an N-terminal chemically modified protein.Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pKadifferences between the e-amino group of the lysine residues and that ofthe a-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

The present invention contemplates use of derivatives which areprokaryote-expressed GDNF, or variants thereof, linked to at least onepolyethylene glycol molecule, as well as use of GDNF, or variantsthereof, attached to one or more polyethylene glycol molecules via anacyl or alkyl linkage.

Pegylation may be carried out by any of the pegylation reactions knownin the art. See, for example: Focus on Growth Factors, 3 (2):4-10, 1992;EP 0 154 316, the disclosure of which is hereby incorporated byreference; EP 0 401 384; and the other publications cited herein thatrelate to pegylation. The pegylation may be carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer).

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol with the GDNF protein or variant. Anyknown or subsequently discovered reactive PEG molecule may be used tocarry out the pegylation of GDNF protein or variant. A preferredactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, "acylation" is contemplated to include without limitation thefollowing types of linkages between the therapeutic protein and a watersoluble polymer such as PEG: amide, carbamate, urethane, and the like.See Bioconjugate Chem., 5:133-140, 1994. Reaction conditions may beselected from any of those known in the pegylation art or thosesubsequently developed, but should avoid conditions of temperature,solvent, and pH that would inactivate the GDNF or variant to bemodified.

Pegylation by acylation will generally result in a poly-pegylated GDNFprotein or variant. Preferably, the connecting linkage will be an amide.Also preferably, the resulting product will be substantially only(e.g., >95%) mono-, di- or tri-pegylated. However, some species withhigher degrees of pegylation may be formed in amounts depending on thespecific reaction conditions used. If desired, more purified pegylatedspecies may be separated from the mixture, particularly unreactedspecies, by standard purification techniques, including, among others,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography and electrophoresis.

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with the GDNF protein or variant in the presence of areducing agent. Pegylation by alkylation can also result inpoly-pegylated GDNF protein or variant. In addition, one can manipulatethe reaction conditions to favor pegylation substantially only at thea-amino group of the N-terminus of the GDNF protein or variant (i.e., amono-pegylated protein). In either case of monopegylation orpolypegylation, the PEG groups are preferably attached to the proteinvia a --CH2--NH-- group. With particular reference to the --CH2-- group,this type of linkage is referred to herein as an "alkyl" linkage.

Derivatization via reductive alkylation to produce a monopegylatedproduct exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available forderivatization. The reaction is performed at a pH which allows one totake advantage of the pKa differences between the e-amino groups of thelysine residues and that of the a-amino group of the N-terminal residueof the protein. By such selective derivatization, attachment of a watersoluble polymer that contains a reactive group such as an aldehyde, to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. In one important aspect, the present inventioncontemplates use of a substantially homogeneous preparation ofmonopolymer/GDNF protein (or variant) conjugate molecules (meaning GDNFprotein or variant to which a polymer molecule has been attachedsubstantially only (i.e., >95%) in a single location). Morespecifically, if polyethylene glycol is used, the present invention alsoencompasses use of pegylated GDNF protein or variant lacking possiblyantigenic linking groups, and having the polyethylene glycol moleculedirectly coupled to the GDNF protein or variant.

Thus, it is contemplated that GDNF protein products to be used inaccordance with the present invention may include pegylated GDNF proteinor variants, wherein the PEG group(s) is (are) attached via acyl oralkyl groups. As discussed above, such products may be mono-pegylated orpoly-pegylated (e.g., containing 2-6, and preferably 2-5, PEG groups).The PEG groups are generally attached to the protein at the a- ore-amino groups of amino acids, but it is also contemplated that the PEGgroups could be attached to any amino group attached to the protein,which is sufficiently reactive to become attached to a PEG group undersuitable reaction conditions.

The polymer molecules used in both the acylation and alkylationapproaches may be selected from among water soluble polymers asdescribed above. The polymer selected should be modified to have asingle reactive group, such as an active ester for acylation or analdehyde for alkylation, preferably, so that the degree ofpolymerization may be controlled as provided for in the present methods.An exemplary reactive PEG aldehyde is polyethylene glycolpropionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxyderivatives thereof (see, U.S. Pat. No. 5,252,714). The polymer may bebranched or unbranched. For the acylation reactions, the polymer(s)selected should have a single reactive ester group. For the presentreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. Generally, the water soluble polymer will notbe selected from naturally-occurring glycosyl residues since these areusually made more conveniently by mammalian recombinant expressionsystems. The polymer may be of any molecular weight, and may be branchedor unbranched.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol. As used herein, polyethylene glycol is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono- (C1-C10) alkoxy- or aryloxy-polyethyleneglycol.

In general, chemical derivatization may be performed under any suitablecondition used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated GDNF proteinor variant will generally comprise the steps of (a) reacting a GDNFprotein or variant with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby the protein becomesattached to one or more PEG groups, and (b) obtaining the reactionproduct(s). In general, the optimal reaction conditions for theacylation reactions will be determined case-by-case based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-pegylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/GDNF protein (or variant) conjugate molecule willgenerally comprise the steps of: (a) reacting a GDNF protein or variantwith a reactive PEG molecule under reductive alkylation conditions, at apH suitable to permit selective modification of the a-amino group at theamino terminus of said GDNF protein or variant; and (b) obtaining thereaction product(s).

For a substantially homogeneous population of mono-polymer/GDNF protein(or variant) conjugate molecules, the reductive alkylation reactionconditions are those which permit the selective attachment of the watersoluble polymer moiety to the N-terminus of GDNF protein or variant.Such reaction conditions generally provide for pKa differences betweenthe lysine amino groups and the a-amino group at the N-terminus (the pKabeing the pH at which 50% of the amino groups are protonated and 50% arenot). The pH also affects the ratio of polymer to protein to be used. Ingeneral, if the pH is lower, a larger excess of polymer to protein willbe desired (i.e., the less reactive the N-terminal a-amino group, themore polymer needed to achieve optimal conditions). If the pH is higher,the polymer:protein ratio need not be as large (i.e., more reactivegroups are available, so fewer polymer molecules are needed). Forpurposes of the present invention, the pH will generally fall within therange of 3-9, preferably 3-6.

Another important consideration is the molecular weight of the polymer.In general, the higher the molecular weight of the polymer, the fewerpolymer molecules may be attached to the protein. Similarly, branchingof the polymer should be taken into account when optimizing theseparameters. Generally, the higher the molecular weight (or the morebranches) the higher the polymer:protein ratio. In general, for thepegylation reactions contemplated herein, the preferred averagemolecular weight is about 2 kDa to about 100 kDa. The preferred averagemolecular weight is about 5 kDa to about 50 kDa, particularly preferablyabout 12 kDa to about 25 kDa. The ratio of water-soluble polymer to GDNFprotein or variant will generally range from 1:1 to 100:1, preferably(for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.

Using the conditions indicated above, reductive alkylation will providefor selective attachment of the polymer to any GDNF protein or varianthaving an a-amino group at the amino terminus, and provide for asubstantially homogenous preparation of monopolymer/GDNF protein (orvariant) conjugate. The term "monopolymer/GDNF protein (or variant)conjugate" is used here to mean a composition comprised of a singlepolymer molecule attached to a molecule of GDNF protein or GDNF variantprotein. The monopolymer/GDNF protein (or variant) conjugate preferablywill have a polymer molecule located at the N-terminus, but not onlysine amino side groups. The preparation will preferably be greaterthan 90% monopolymer/GDNF protein (or variant) conjugate, and morepreferably greater than 95% monopolymer/GDNF protein (or variant)conjugate, with the remainder of observable molecules being unreacted(i.e., protein lacking the polymer moiety).

For the present reductive alkylation, the reducing agent should bestable in aqueous solution and preferably be able to reduce only theSchiff base formed in the initial process of reductive alkylation.Preferred reducing agents may be selected from sodium borohydride,sodium cyanoborohydride, dimethylamine borane, trimethylamine borane andpyridine borane. A particularly preferred reducing agent is sodiumcyanoborohydride. Other reaction parameters, such as solvent, reactiontimes, temperatures, etc., and means of purification of products, can bedetermined case-by-case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein).

C. GDNF Protein Product Pharmaceutical Compositions

GDNF protein product pharmaceutical compositions typically include atherapeutically effective amount of a GDNF protein product in admixturewith one or more pharmaceutically and physiologically acceptableformulation materials. Suitable formulation materials include, but arenot limited to, antioxidants, preservatives, coloring, flavoring anddiluting agents, emulsifying agents, suspending agents, solvents,fillers, bulking agents, buffers, delivery vehicles, diluents,excipients and/or pharmaceutical adjuvants. For example, a suitablevehicle may be water for injection, physiological saline solution, orartificial perilymph, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles.

The primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain otherpharmaceutically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the vehicle maycontain still other pharmaceutically-acceptable excipients for modifyingor maintaining the rate of release of GDNF protein product, or forpromoting the absorption or penetration of GDNF protein product acrossthe tympanic membrane. Such excipients are those substances usually andcustomarily employed to formulate dosages for middle-ear administrationin either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready to use form or in a form, e.g., lyophilized, requiringreconstitution prior to administration.

The optimal pharmaceutical formulations will be determined by oneskilled in the art depending upon considerations such as the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present GDNF proteins, variants and derivatives.

Other effective administration forms, such as middle-ear slow-releaseformulations, inhalant mists, or orally active formulations are alsoenvisioned. For example, in a sustained release formulation, the GDNFprotein product may be bound to or incorporated into particulatepreparations of polymeric compounds (such as polylactic acid,polyglycolic acid, etc.) or liposomes. Hylauronic acid may also be used,and this may have the effect of promoting sustained duration in thecirculation. The GDNF protein product pharmaceutical composition alsomay be formulated for middle-ear administration, e.g., by tympanicmembrane infusion or injection, and may also include slow-release orsustained circulation formulations. Such middle-ear administeredtherapeutic compositions are typically in the form of a pyrogen-free,middle-ear acceptable aqueous solution comprising the GDNF proteinproduct in a pharmaceutically acceptable vehicle. One preferred vehicleis sterile distilled water.

It is also contemplated that certain formulations containing GDNFprotein product are to be administered orally. GDNF protein productwhich is administered in this fashion may be encapsulated and may beformulated with or without those carriers customarily used in thecompounding of solid dosage forms. The capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional excipients may beincluded to facilitate absorption of GDNF protein product. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

The formulation of topical ear preparations, including middle-earsolutions, suspensions and ointments is well known to those skilled inthe art (see Remington's Pharmaceutical Sciences, 18th Edition, Chapter86, pages 1581-1592, Mack Publishing Company, 1990). Other modes ofadministration are available, including injections to the middle ear,and methods and means for producing middle-ear preparations suitable forsuch modes of administration are also well known.

As used in this application, "middle-ear" refers to the space betweenthe tympanic membrane and the inner ear. This location is external toall inner ear tissue and an invasive procedure might not be required toaccess this region if a formulation will be developed so that the GDNFwill penetrate through the tympanic membrane. Otherwise, the materialwill be introduced to the middle ear by injection through the tympanicmembrane or, in case repeated administrations are needed, a hole will bemade in the tympanic membrane. Examples of such systems include insertsand "topically" applied drops, gels or ointments which may be used todeliver therapeutic material to these regions. An opening in thetymapanic membrane is a very frequent procedure done on a office-visitbasis, in cases such as infections of the middle ear (usually inchildren). The opening closes spontaneously after a few days.

In the presently described use of GDNF protein product of the treatmentof inner ear disease or injury it is also advantageous that a topicallyapplied formulation include an agent to promote the penetration ortransport of the therapeutic agent into the middle and inner ear. Suchagents are known in the art. For example, Ke et al., U.S. Pat. No.5,221,696 disclose the use of materials to enhance the penetration ofophthalmic preparations through the cornea.

Inner-ear systems are those systems which are suitable for use in anytissue compartment within, between or around the tissue layers of theinner-ear, such as the cochlea and vestibular organ. These locationsinclude the different structures of the cochlea such as the striavascularis, Reissner's membrane. organ of Corti, spiral ligament and thecochlear neurons. An invasive procedure might not be required to accessthose structures since it has been shown that protein do penetrate themembrane of the round window into the perylimph of the inner ear. Aparticularly suitable vehicle for introducing GDNF into the inner ear bypenetration through the round window membrane is artificial perylimph.This solution consists of 10.00 mM D-glucose, 1,5 mM CaCl, 1.5 mM MgClin a 1.0% solution of Dulbeco's phosphate-buffered saline in deionizedwater at 280-300 mOsm and pH of 7.2. Yet another preparation may involvethe formulation of the GDNF protein product with an agent, such asinjectable microspheres or liposomes into the middle ear, that providesfor the slow or sustained release of the protein which may then bedelivered as a depot injection. Other suitable means for the inner-earintroduction of GDNF protein product includes, implantable drug deliverydevices or which contain the GDNF protein product, and acochlear-implant with a tunnel through, so GDNF can be continuouslydelivered through it to the inner ear.

The ear-treatment preparations of the present invention, particularlytopical preparations, may include other components, for examplemiddle-ear acceptable preservatives, tonicity agents, cosolvents,complexing agents, buffering agents, antimicrobials, antioxidants andsurfactants, as are well known in the art. For example, suitabletonicity enhancing agents include alkali metal halides (preferablysodium or potassium chloride), mannitol, sorbitol and the like.Sufficient tonicity enhancing agent is advantageously added so that theformulation to be instilled into the ear is compatible with theosmolarity of the endo- and perilymph. Suitable preservatives include,but are not limited to, benzalkonium chloride, thimerosal, phenethylalcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid andthe like. Hydrogen peroxide may also be used as preservative. Suitablecosolvents include, but are not limited to, glycerin, propylene glycoland polyethylene glycol. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. The buffers can be conventional bufferssuch as borate, citrate, phosphate, bicarbonate, or Tris-HCl.

The formulation components are present in concentration that areacceptable to the middle or inner ear site of administration. Forexample, buffers are used to maintain the composition at physiologicalpH or at slightly lower pH, typically within a pH range of from about 5to about 8.

Additional formulation components may include materials which providefor the prolonged the residence of the middle ear administeredtherapeutic agent so as to maximize the topical contact and promoteabsorbtion through the round window membrane. Suitable materials includepolymers or gel forming materials which provide for increased viscosityof the middle-ear preparation. The suitability of the formulations ofthe instant invention for controlled release (e.g., sustained andprolonged delivery) of an inner-ear treating agent can be determined byvarious procedures known in the art. Yet another ear preparation mayinvolve an effective quantity of GDNF protein product in a mixture withnon-toxic middle-ear treatment acceptable excipients which are suitablefor the manufacture of tablets. By dissolving the tablets in sterilewater, or other appropriate vehicle, middle-ear treatment solutions canbe prepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia.

Administration/Delivery of GDNF Protein Product

The GDNF protein product may be administered parenterally via asubcutaneous, intramuscular, intravenous, transpulmonary, transdermal,intrathecal or intracerebral route. For the treatment of inner-earconditions, the GDNF protein product may be administered into themiddle-ear (or directly into the inner-ear, especially in cases whereinvasive procedure is already in place), by topical application,inserts, injection, implants, cell therapy or gene therapy. For example,slow-releasing implants containing the neurotrophic factor embedded in abiodegradable polymer matrix can deliver GDNF protein product. GDNFprotein product may be administered extracerebrally in a form that hasbeen modified chemically or packaged so that it passes the blood-brainbarrier, or it may be administered in connection with one or more agentscapable of promoting penetration of GDNF protein product across thebarrier. Similarly, the GDNF protein product may be administered in themiddle or inner ear, or it may be administered on top of the tympanicmembrane in connection with one or more agents capable of promotingpenetration or transport of GDNF protein product across the membranes ofthe ear. The frequency of dosing will depend on the pharmacokineticparameters of the GDNF protein product as formulated, and the route ofadministration.

The specific dose may be calculated according to considerations of bodyweight, body surface area or organ size. Further refinement of thecalculations necessary to determine the appropriate dosage for treatmentinvolving each of the above mentioned formulations is routinely made bythose of ordinary skill in the art and is within the ambit of tasksroutinely performed, especially in light of the dosage information andassays disclosed herein. Appropriate dosages may be ascertained throughuse of the established assays for determining dosages utilized inconjunction with appropriate dose-response data. It will be appreciatedby those skilled in the art that the dosage used in inner-earadministered formulations will be minuscule as compared to that used ina systemic injection or oral administration.

The final dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g., the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. As studies are conducted, further information will emergeregarding the appropriate dosage levels for the treatment of variousdiseases and conditions.

It is envisioned that the continuous administration or sustaineddelivery of GDNF may be advantageous for a given treatment. Whilecontinuous administration may be accomplished via a mechanical means,such as with an infusion pump, it is contemplated that other modes ofcontinuous or near continuous administration may be practiced. Forexample, chemical derivatization or encapsulation may result insustained release forms of the protein which have the effect ofcontinuous presence, in predictable amounts, based on a determineddosage regimen. Thus, GDNF protein products include proteins derivatizedor otherwise formulated to effectuate such continuous administration.

GDNF protein product cell therapy, e.g., middle- or inner earimplantation of cells producing GDNF protein product, is alsocontemplated. This embodiment would involve implanting into patientscells capable of synthesizing and secreting a biologically active formof GDNF protein product. Such GDNF protein product-producing cells maybe cells that are natural producers of GDNF protein product (analogousto B49 glioblastoma cells) or may be recombinant cells whose ability toproduce GDNF protein product has been augmented by transformation with agene encoding the desired GDNF protein product in a vector suitable forpromoting its expression and secretion. In order to minimize a potentialimmunological reaction in patients being administered GDNF proteinproduct of a foreign species, it is preferred that the natural cellsproducing GDNF protein product be of human origin and produce human GDNFprotein product. Likewise, it is preferred that the recombinant cellsproducing GDNF protein product be transformed with an expression vectorcontaining a gene encoding a human GDNF protein product. Implanted cellsmay be encapsulated to avoid infiltration of surrounding tissue. Humanor non-human animal cells may be implanted in patients in biocompatible,semipermeable polymeric enclosures or membranes that allow release ofGDNF protein product, but that prevent destruction of the cells by thepatient's immune system or by other detrimental factors from thesurrounding tissue. Such an implant, for example, may be attached to theround-window membrane of the middle-ear to produce and release GDNFprotein product directly into the perilymph.

It is also contemplated that the patient's own cells may be transformedex vivo to produce GDNF protein product and would be directly implantedwithout encapsulation. For example, organ of Corti supporting cells maybe retrieved, the cells cultured and transformed with an appropriatevector and transplanted back into the patient's inner ear where theywould produce and release the desired GDNF protein or GDNF proteinvariant.

GDNF protein product gene therapy in vivo is also envisioned, byintroducing the gene coding for GDNF protein product into targeted innerear cells via local injection of a nucleic acid construct or otherappropriate delivery vectors. (Hefti, J. Neurobiol., 25:1418-1435,1994). For example, a nucleic acid sequence encoding a GDNF proteinproduct may be contained in an adeno-associated virus vector oradenovirus vector for delivery to the inner ear cells. Alternative viralvectors include, but are not limited to, retrovirus, herpes simplexvirus and papilloma virus vectors. Physical transfer, either in vivo orex vivo as appropriate, may also be achieved by liposome-mediatedtransfer, direct injection (naked DNA), receptor-mediated transfer(ligand-DNA complex), electroporation, calcium phosphate precipitationor microparticle bombardment (gene gun).

The methodology for the membrane encapsulation of living cells isfamiliar to those of ordinary skill in the art, and the preparation ofthe encapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538, 5,011,472, and 5,106,627, each of which is specificallyincorporated herein by reference. A system for encapsulating livingcells is described in PCT Application WO 91/10425 of Aebischer et al.,specifically incorporated herein by reference. See also, PCP ApplicationWO 91/10470 of Aebischer et al., Winn et al., Exper. Neurol.,113:322-329, 1991, Aebischer et al., Exper. Neurol., 111:269-275, 1991;Tresco et al., ASAIO, 38:17-23, 1992, each of which is specificallyincorporated herein by reference. Techniques for formulating a varietyof other sustained-or controlled-delivery means, such as liposomecarriers, bio-erodible particles or beads and depot injections, are alsoknown to those skilled in the art.

It should be noted that the GDNF protein product formulations describedherein may be used for veterinary as well as human applications and thatthe term "patient" should not be construed in a limiting manner. In thecase of veterinary applications, the dosage ranges should be the same asspecified above.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples. Example 1addresses the effects of GDNF protein product administration on haircells in a Cochlea explant culture system. Example 2 addresses theeffects of GDNF protein product administration on spiral ganglionneurons, in a dissociated cell culture generated from cochlea. Theresults of the organ of Corti explant cultures studies and that of theadult rat spiral ganglion neuronal cultures demonstrated that GDNFprotein product has neurotrophic and protective activity for theauditory neurons and the hair cells of the organ of Corti againstototoxins, which were not previously known to be GDNF-responsive.

EXAMPLES Example 1 GDNF Protein Product Protects Cochlear Hair CellsAgainst Ototoxicity

MATERIALS

The materials used in the following Example were obtained as follows.

Organ of Corti dissecting solution:

Dulbecco's Phosphate Buffered Saline (PBS; 1×, without calcium chloride,without magnesium chloride. Cat. #14190-136, Gibco BRL), containing 1.5g/L D-Glucose (Dextrose. Cat. #15023-021, Gibco BRL).

Organ of Corti explant culture Medium

1. High glucose Dulbecco's Modified Eagle Medium (DMEM; 1×, withL-glutamine, without Sodium Pyruvate. Cat. #11965-084, Gibco BRL)

2. 0.15 g/100 ml of D-Glucose (Dextrose. Cat. #15023-021, Gibco BRL)

3. 1% N-2 Supplement (100 ×, Cat. #17502-030, Gibco BRL)

4. 100 Units/ml of Penicillin G, Potassium (Penicillin; Cat. #21840-020,Gibco BRL)

METHODS

Preparation of Medium

DMEM was supplemented with 1% N-2 supplement, and D-glucose was added toa final concentration of 1.5 g/L. Penicillin was added at 100 Units/ml.After mixing, the medium was filtered and kept at 4° C. The medium wasprepared fresh just before use in order to minimize inter-experimentalvariations. Plastic pipettes and containers were used throughout tominimize protein adsorption.

GDNF Protein Product Solutions

Purified human recombinant GDNF protein products were prepared as 1mg/ml solutions in D-PBS (phosphate buffered saline prepared withdistilled water) containing five percent bovine serum albumin. Thesolutions were stored at -85° C. in aliquots. Serial dilutions (0.1; 1;10; 25; 50 ng/ml in normal culture medium) were prepared in 96microplates. Ten microliters of ten-fold concentrated GDNF proteinproduct solutions were added to Organ of Corti explant cultures mediumcontaining ototoxins or not (control)(90 μl). Control cultures receivednormal medium(10 μl). The GDNF protein product treatments were initiatedon day of plating. On the second day, media were exchanged into mediacontaining the ototoxins alone, together with GDNF or without both(control).

Dissecting tools and culture dishes 1. The 4" and 5" dissecting forcepsand 4" dissecting scissors were from Roboz Surgical, Washington, DC. 2.Falcon sterile 96-well microplates (Flat Bottom. Cat. #3072), tissueculture plastic ware and polypropylene centrifuge tubes were fromBeckton-Dickinson, Lincoln Park, N.J.

Ototoxins and Related Reagents

1. Neomycin solution (Cat. #N1142, Sigma. St. Louis, Mo.), used at finalconcentration of 0.6 mM (A fresh solution was made each experiment byadding 90 μl of 1 mg/ml neomycin and to 1410 μl medium).

2. Cisplatin (Platinol-AQ. Cat. #NDC 0015-3220-22, Bristol-Myers SquibbLaboratories, Princeton, N.J.). Used at a final concentration of 35μg/ml (a fresh solution was prepared each experiment by adding 52.5 μlof 1 mg/ml cisplatin to 1447.5 μl medium)

3. Triton X-100 (t-Octylphenoxypoly-ethoxyethanol. Cat. #X-100, Sigma.St. Louis, Mo.)

4. Phalloidin (FITC Labeled. Cat. #P-5282, Sigma. St. Louis, Mo.)

5. Vectashield (Mounting Medium, Cat. #H-1000, Vector, Burlingame,Calif.)

Preparation of Rat Organ of Corti explant

Organ of Corti explants were obtained from P3-P4 Wistar rats. Rats weredecapitated, the lower jaw was cut out and skin removed. The temporalbone was collected in dissection solution, the otic capsule exposed andthe bony-cartilaginous cochlear capsule was carefully separated from thetemporal bone. Freed cochlea were transferred to another Petri dish withdissection solution for further dissection. Intact organs of Corti wereobtained by using a fine forceps to hold central VIII nerve tissue andremove it out, then the stria vascular membrane was carefully strippedoff, starting from the apex or base. The organ of Corti then was thentransferred to a 35-mm diameter Petri dish containing cold PBSsupplemented with glucose and ready to be cultured.

Cochlea explant culture Procedure

Cochlea explants were cultured in uncoated 96 microplates. A singleorgan of Corti was placed in a well and was kept floating in the medium.Explants were kept in normal medium for 24 hours (90 μl/well). GDNFprotein solution (10 μl) was added to the `treated` cultures and 10 μlmedium was added to controls. After 24 hours of incubation, the mediawere changed and the explants were exposed to ototoxin-containing medium(90 μl), with GDNF protein solution (10 μl) or without (control). Thecultures were incubated for an additional 3 days. The explants were thenfixed with 4% paraformaldehyde in 0.1M D-PBS for 30 minutes at roomtemperature and processed for immunostaining.

FITC-phalloidin staining of hair cells

To identify and count hair cells in the organ of Corti, a directimmunostaining method was used to label the actin present naturally inthe stereocilia bundles of the hair cells. The explants were washedthree times with D-PBS (200 μl per well) and permeabilized with 1%Triton X-100 in D-PBS for 15 minutes at room temperature. After threewashes in D-PBS, the explants were incubated with FITC-labeledPhalloidin (1:60 from stock, 50 μl/well) for 45 minutes at roomtemperature. The plates were covered with aluminum foil as thePhalloidin is light sensitive. After three more washes with D-PBS, thelabeled explants were placed in a drop of glycerol on a microscopeslide, covered with a glass coverslip and sealed with nail polish. Theexplants were observed under a Nikon Diaphot-300 inverted fluorescencemicroscope, using FITC filters and fluorescence optics.

Determination of hair cell number

For each experimental point, 2 to 4 cochlea were used. In each cochlea,the number of hair cells was counted in 2-3 sections, 175 μm in lengtheach. Only the sections in the middle turn of the cochlea were analyzed.Each experiment was repeated several times. The numbers of hair cells incontrol and cisplatin- or neomycin-treated cultures was generated fromanalyzing 40 cochlea per point.

RESULTS

Hair cells in the floating explant cultures did not die during theexperiment period of four days. Thus, the number of phalloidin-stainedcells present at the end of the 4 days experiment period, in the absenceof ototoxins and treatments, was 105.4±6.9 (n=28). Ototoxins added tothe explants on the second day post plating caused significant loss inhair cell number found after 4 days in vitro. Exposure to 35 μg/mlcisplatin 24 hours after plating caused a loss of about 80 percent ofthe hair cells: only 21.2%±6.0 (n=40) of the initial number of haircells survived (Table 2) and after exposure to 0.8 mM neomycin, only7.4%±4.7 (n=43) of the hair cells survived (Table 2).

                  TABLE 2                                                         ______________________________________                                        Effect of GDNF on cochlear hair cells exposed to cisplatin                                Hair Cell Survival (% of untreated)                                             GDNF added    GDNF added 24                                     Treatment     at time of plating                                                                          hours after plating                               ______________________________________                                        Cisplatin alone (35 μg/ml)                                                               21.3 ± 4.0 (n = 30)                                                                      21.3 ± 4.0 (n = 30)                            Cisplatin     35.2 ± 5.4 (n = 5)*                                                                      ND                                                + GDNF, 0.05 ng/ml                                                            Cisplatin     39.6 ± 10.5 (n = 17)*                                                                    37.8 ± 11.8 (n = 5)*                           + GDNF, 0.1 ng/ml                                                             Cisplatin     46.7 ± 10.8 (n = 20)*                                                                    51.0 ± 8.0 (n = 4)*                            + GDNF, 1 ng/ml                                                               Cisplatin     46.7 ± 7.7 (n = 16)*                                                                     49.7 + 4.6 (n = 5)*                               + GDNF, 10 ng/ml                                                              Cisplatin     ND            45.0 ± 12.0 (n = 3)*                           + GDNF, 25 ng/ml                                                              Cisplatin     46.8 ± 10.5 (n = 13)*                                                                    ND                                                + GDNF, 50 ng/ml                                                              ______________________________________                                         GDNF was introduced to the explant cultures either on the day of plating      or 24 hours after plating. Cisplatin (35 μg/ml) was added 24 hours         after plating, and the cultures were incubated for an additional 3 days.      The hair cells were stained with FITCphalloidin. The number of hair cells     was counted in the middle turn of the cochlea in 2-3 sections of 175 μ     each. The results are expressed as the percentage of hair cells present i     untreated cultures after 4 days in vitro (105.4 ± 6.9; n = 28). Each       number is the mean ± SD of n cochleas.                                     *Significantly different from cisplatin alone at p < 0.05 (tTest)             ND: not determined                                                       

There was a marked difference in the morphology of the organs of Cortibetween these two treatments: while the treatment with neomycin resultedin almost complete loss of hair cells, those that were spared were stillorganized in the typical four row structure (3 rows of outer hair cellsand one row of inner hair cells). Cisplatin treatment, on the otherhand, caused a marked disruption of the four-row-structure and thesurviving cells were randomly located.

In cultures that received GDNF at the time of plating (pretreatment), asignificant number of hair cells survived the 3-day exposure toototoxins (from day 2 to day 4). In cultures exposed to cisplatin,treatment with GDNF concentrations as low as 0.05 ng/ml caused anincrease in surviving hair cells from 21% (untreated cultures) to 35%.Maximal protective activity was reached with 0.1 ng/ml GDNF (41%survival) (Table 3). In cultures exposed to neomycin, GDNF at 0.1 ng/mlincreased the number of hair cells from 7% to 22%; maximal GDNF activity(37% survival) was seen with 10 ng/ml GDNF (Table 3). GDNF treatmentpreserved the four-raw morphology in neomycin-treated cultures, but didnot prevent its disruption by cisplatin.

To test further the ability of GDNF to rescue hair cells fromototoxicity, a set of experiments was performed in which GDNF was added24 hours after plating, at the same time as the cultures were exposed tothe toxins (co-treatment). The results indicate that under thisexperimental paradigm GDNF is capable of rescuing hair cells to the sameextent as when given prior to the exposure to the toxins (Tables 2 and3)

                  TABLE 3                                                         ______________________________________                                        Effect of GDNF on cochlear hair cells exposed to neomycin                                  Hair Cell Survival (% of untreated)                                             GDNF added   GDNF added                                        Treatment      at time of plating                                                                         24 hr after plating                               ______________________________________                                        Neomycin alone(0.8 mM)                                                                        7.1 ± 4.2 (n = 42)                                                                      7.1 + 4.2 (n = 42)                               Neomycin       19.5 ± 7.5 (n = 6)*                                                                     23.0 ± 6.2 (n = 3)*                            + GDNF, 0.05 ng/ml                                                            Neomycin       22.0 ± 4.1 (n = 13)*                                                                    27.0 ± 14.7 (n = 3)*                           + GDNF, 0.1 ng/ml                                                             Neomycin       28.2 ± 6.1 (n = 19)*                                                                    26.2 ± 6.4. (n = 4)*                           + GDNF, 1 ng/ml                                                               Neomycin       37.4 ± 4.8 (n = 11)*                                                                    ND                                                + GDNF, 10 ng/ml                                                              Neomycin       34.4 + 5.3 (n = 7)*                                                                        ND                                                + GDNF, 50 ng/ml                                                              ______________________________________                                         GDNF was introduced to the explant cultures either on the day of plating      (pretreatment) or 24 hours af ter plating (cotreatment). Neomycin (0.8M)      was added 24 hours after plating, and the cultures were incubated for an      additional 3 days. The hair cells were stained with FITCphalloidin. The       number of hair cells was counted in the middle turn of the cochlea in 2-3     sections of 175 μm each. The results are expressed as the percentage o     hair cells present in untreated cultures after 4 days in vitro (105.4 .+-     6.9; n = 28). Each number is the mean ± SD of n cochleas.                  *Significantly different from neomycin alone at p < 0.05 (tTest)              ND: not determined                                                       

Example 2 GDNF Protein Product Promotes Survival of Inner Ear AuditoryNeurons (Spiral Ganglion Neurons) and Protects Them Against Ototoxins

MATERIALS

The materials used in the following Example were obtained as follows.

Cell Culture Media

High glucose Dulbecco's Modified Eagle's Medium (DMEM; #11965-092),Ham's F12 medium (F12;#11765-021), B27 medium supplement (#17504-010),penicillin/streptomycin (#15070-014), L-glutamine (#25030-016),Dulbecco's phosphate-buffered saline (D-PBS; #14190-052), mouse laminin(#23017-015), bovine serum albumin and fractionV (#110-18-017) were allfrom GIBCO/BRL, Grand Island, N.Y. Heat-inactivated horse serum was fromHyClone, Logan, Utah. Poly-L-ornithine hydrobromide (P-3655), bovineinsulin (I-5500), human transferrin (T-2252), putrescine (P-6024),progesterone (P-6149) and sodium selenite (S-9133) were all from SigmaChemical Company, Saint-Louis, Mo. Papain, deoxyribonuclease I (DNAase)and ovalbumin (Papain dissociation system) were from WorthingtonBiochemicals, Freehold, N.J. Falcon sterile 96-well microplates (#3072),tissue culture plastic ware and polypropylene centrifuge tubes were fromBeckton-Dickinson, Oxnard, Calif. Nitex 20 μm nylon mesh (#460) was fromTetko, Elmsford, N.Y. The 4" dissecting forceps and 4" dissectingscissors were from Roboz Surgical, Washington, D.C.

Antibodies and Related Reagents

Neuronal Specific Enolase (NSE) rabbit polyclonal antibody, was fromChemicon (#AB951), biotinylated goat anti-rabbit IgG (#BA-1000) andperoxidase-conjugated avidin/biotin complex (ABC Elite; kit PK-6100)were from Vector Laboratories, Burlingame, Calif. 3',3'-diaminobenzidine was from Cappel Laboratories, West Chester, Pa.Superblock blocking buffer in PBS (#37515) was from Pierce, Rockford,Ill. Triton X-100 (X100), Nonidet P-40 (N6507) and hydrogen peroxide(30%, v/v; H1009) were from Sigma. All other reagents were obtained fromSigma Chemical Company (Saint-Louis, Mo.), unless otherwise specified.

Ototoxins

Cisplatin (Platinol-AQ, #NDC 0015-3220-22) was fromBristol-Myers-Squibb, Princeton, N.J., sodium salicylate was from J.T.Baker, Phillipsburg, N.J. (#3872-01) and neomycin was from Sigma(#N1142).

METHODS

Preparation of Media

A basal medium was prepared as a 1:1 mixture of DMEM and F12 medium, andwas supplemented with B27 medium supplement added as a 50-foldconcentrated stock solution. The B27 medium supplement consists ofbiotin, L-carnitine, corticosterone, ethanolamine, D(+)- galactose,reduced glutathione, linoleic acid, linolenic acid, progesterone,putrescine, retinyl acetate, selenium, T3 (triodo-1-thyronine,DL-alpha-tocopherol; vitamin E), DL-alpha-tocopherol acetate, bovineserum albumin, catalase, insulin, superoxide dismutase and transferrin.L-glutamine was added at a final concentration of about 2 mM, penicillinat about 100 IU/l, and streptomycin at about 100 mg/l. Heat-inactivatedhorse serum was added to a final concentration of about 2.5 percent,D-glucose was added to a final concentration of about 5 g/l, HEPESbuffering agent was added to a final concentration of about 20 mM,bovine insulin was added to a final concentration of about 2.5 mg/ml,and human transferrin was added to a final concentration of about 0.1mg/ml. After mixing, the pH was adjusted to about 7.3 and the medium waskept at 40° C. The media were prepared fresh just before use in order tominimize inter-experimental variations. Plastic pipettes and containerswere used throughout to minimize protein adsorption.

GDNF Protein Product Solutions

Purified human recombinant GDNF protein products were prepared as 1mg/ml solutions in D-PBS (phosphate-buffered saline prepared withdistilled water) containing five percent bovine serum albumin. Thesolutions were stored at -85° C. in aliquots. Serial dilutions wereprepared in 96-well microplates. Ten microliters of ten-foldconcentrated GDNF protein product solutions were added to cell culturescontaining culture medium (90 μl). Control cultures received D-PBS with5 percent albumin (10 μl). The GDNF protein product treatments wereadded to the cultures one hour after cells were seeded or 24 hourslater, alone or together with the ototoxins.

Ototoxins preparations

Neomycin was added straight from the stock solution (about 10³¹ 3 M) at10 μl per well to result in a final concentration of about 10³¹ 4 M.Cisplatin was diluted with culture medium from the stock solution (1mg/ml) to a solution of 20 μg/ml and added at 10 μl per well, to resultin a final concentration of 2 μg/ml. Sodium salicylate was prepared frompowder to a stock solution of 1M in PBS and further diluted in theculture medium to 100 mM, which resulted in a 10 mM final concentrationwhen added at 10 μl/well to the culture.

Culture Substratum

To encourage optimal attachment of spiral ganglion cells on substratumand neurite outgrowth, the microtiter plate surfaces (the culturesubstratum) were modified by sequential coating with poly-L-ornithinefollowed by laminin in accordance with the following procedure. Theplate surfaces were completely covered with a 0.1 mg/ml sterile solutionof polyornithine in 0.1M boric acid (pH 8.4) for at least one hour atroom temperature, followed by a sterile wash with Super-Q water. Thewater wash was then aspirated and a 10 μg/ml solution of mouse lamininin PBS was added and incubated at 37° C. for two hours. These procedureswere conducted just before using the plates in order to ensurereproducibility of the results.

Preparation of Rat Spiral Ganglion Cell Cultures

Three- to four-week-old Wistar rats (obtained from Jackson Laboratories,Bar Harbor, Me.) were injected with an overdose of the followingsolution (ketamine (100 mg/ml); Xylazine (20 mg/ml) and AcopromazineMaleate 910 mg/ml) at 3:3:1 proportions), killed by decapitation and thetemporal bone with the cochlea were dissected out and transferredsterilely into PBS with 1.5 g/L glucose on ice. A maximum of 30 cochleawere processed per experiment. The cochlea were opened and the organ ofCorti with the bony modiolus was collected into 50 ml sterile tubecontaining 5 ml dissociation medium (120 units papain and 2000 unitsDNAase in HBSS). The tissue was incubated for 30 minutes at about 37° C.on a rotary platform shaker at about 200 rpm and then the dissociationsolution was replaced with a fresh one and the incubation resumed foranother 30 min. The cells were then dispersed by trituration throughfire-polished Pasteur pipettes, sieved through a 40 μm Nitex nylon meshto discard undissociated tissue, and centrifuged for five minutes at200×g using an IEC clinical centrifuge. The resulting cell pellet wasresuspended into HBSS containing ovalbumin and about 500 units DNAase,layered on top of a four percent ovalbumin solution (in HBSS) andcentrifuged for about 6 minutes at 500×g. The final pellet wasresuspended into about 6 ml of the culturing medium and seeded at 90μl/well in the precoated plates.

Immunohistochemistry of spiral ganglion cells

Spiral ganglion neurons were identified by immunohistochemical stainingfor neuronal specific enolase (NSE). Cultures of spiral ganglion cellswere fixed for about 10 minutes at room temperature with eight percentparaformaldehyde in D-PBS, pH 7.4, added at 100 μl/well to the culturemedium and then replaced by 100 μl of four percent paraformaldehyde foradditional 10 minutes, followed by three washes in D-PBS (200 μl per6-mm well). The fixed cultures were then incubated in Superblockblocking buffer in PBS, containing one percent Nonidet P-40 to increasethe penetration of the antibody. The rabbit polyclonal anti-NSEantibodies (Chemicon) were then applied at a dilution of 1:6000 in thesame buffer, and the cultures were incubated for two hours at 37° C. ona rotary shaker. After three washes with D-PBS, the spiral ganglioncell-bound antibodies were detected using goat-anti-rabbit biotinylatedIgG (Vectastain kit from Vector Laboratories, Burlingame, Calif.) atabout a 1:300 dilution. The secondary antibody was incubated with thecells for about one hour at 37° C., the cells were then washed threetimes with D-PBS. The secondary antibody was then labeled with anavidin-biotin-peroxidase complex diluted at 1:300, and the cells wereincubated for about 60 minutes at 37° C. After three more washes withD-PBS, the labeled cell cultures were reacted for 5 minutes in asolution of 0.1M Tris-HCl, pH 7.4, containing 0.04% 3',3'-diaminobenzidine-(HCl)4, 0.06 percent NiCl2 and 0.02 percent hydrogenperoxide.

Determining spiral Ganglion Cell Survival

After various times in culture (24 hours, 3 days and 4 days), rat spiralganglion cell cultures were fixed, processed and immunostained for NSEas described above, and the cultures were then examined withbright-light optics at 200× magnification. All the NSE-positive neuronspresent in a 6-mm well were counted. Viable spiral ganglion cells werecharacterized as having a round body of size ranging from 15-40 μm andbearing neuritic processes. Spiral ganglion cells showing signs ofdegeneration, such as having irregular, vacuolated perikarya orfragmented neurites, were excluded from the counts (most of thedegenerating spiral ganglion cells, however, detached from the culturesubstratum). Cell numbers were expressed either as cells/6-mm well or asthe fold-change relative to control cell density.

RESULTS

Cultures of rat spiral ganglion neurons were used to demonstrate theeffect of GDNF protein product on survival and protection againstototoxins. The spiral ganglion cells were obtained from three to four-week old rat cochlea. The dissociated cells were then seeded intopolyornithine-laminin-coated microplates at a density of about 1 cochleaper 2 wells in DMEM/F12 supplemented with B27 medium supplement, 2.5percent heat-inactivated horse serum, D-glucose, HEPES, insulin andtransferrin. The cultures consisted of a mixture of neurons andnon-neuronal cells. However, the only neurons present were spiralganglion neurons and were identified by the presence of NSEimmunoreactivity. At the concentration seeded(1 dissociated organ ofCorti into 2 wells), the number of NSE-positive cells per well 24 hoursafter seeding was 127±17 (n=7) under control conditions (no addedtreatments). At the end of the experiment, 4 days after seeding, thenumber of the neurons per well was reduced to 64±4.7, which represents50.5%±3.7 of the number present after 24 hours in vitro, under controlconditions.

The effect of GDNF protein product administration was assessed on thesurvival and morphological maturation of cultured rat spiral ganglionneurons, as well as on their ability to resist the toxic effect of aknown ototoxin such as cisplatin. Cultures of spiral ganglion cells weretreated 24 hours after seeding with human recombinant GDNF proteinproduct (ranging from 50 ng/ml to 0.1 ng/ml) alone, or in combinationwith cisplatin (35 μg/ml). Twenty four hours after seeding, there was nodifference in the number of auditory neurons between control culturesand those treated with GDNF at 1 ng/ml and 10 ng/ml (127±17; 126±24 and125±19 neurons/well respectively). After an additional period of 3 days,treatment with GDNF at a concentration of 1 ng/ml did not result in asignificant increase in neuronal cell number. There was however, amarked trophic effect: the neuronal soma were larger and fibers longerand more elaborate than in control cultures. In cultures treated with 10ng/ml GDNF, about 72% of the neurons present after 24 hours survived,representing a 44% increase over control cultures (Table 4). The trophiceffect was even stronger than in cultures treated with 1 ng/ml GDNF.

                  TABLE 4                                                         ______________________________________                                        Effect of GDNF on spiral ganglion neuron survival                                         Spiral Ganglion Neuron Survival                                               (% of initial number after 24 hours)                              Treatments    None        Cisplatin (5 μg/ml)                              ______________________________________                                        None          48.5 ± 4.5                                                                              6.1 ± 1.2                                                     (n = 9)     (n = 3)                                             GDNF, 10 ng/ml                                                                              71.6 + 7.4**                                                                              32.8 ± 1.0*                                                    (n = 5)     (n = 3)                                             ______________________________________                                         GDNF and cisplatin were added to dissociated spiral ganglion neuron           cultures 24 hours after plating. The cultures were incubated for an           additional 3 days and the number of neurons was determined by counting        NSEimmunoreactive cells. Neuronal cell numbers are expressed as the           percentage of neurons present after 24 hours in vitro. Each result is the     mean ± SD of n cultures.                                                   *Significantly different from cisplatin alone at p < 0.05 (tTest)             **Significantly different from untreated control at p < 0.05 (tTest)     

GDNF also protected spiral ganglion neurons from cisplatin toxicity(Table 4). Exposure of cultures to 5 μg/ml cisplatin 24 hours afterseeding resulted in the loss of about 94% of the initial number (at 24hours) of the neurons after 4 days in culture. When GDNF was addedtogether with the cisplatin, the number of neurons found after 4 dayswas significantly higher. This protective effect of GDNF wasdose-dependent: 20.1±5.1; 27.5±3.2 and 32.8±1.0 of the neurons presentat the beginning of the toxic treatment were found with GDNFconcentrations of 1 ng/ml, 10 ng/ml and 25 ng/ml respectively (data notshown). These results indicate that about 63 percent of the neurons thatrespond to GDNF (about 44% of the spiral ganglion neuron population) canalso be protected against cisplatin toxicity.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description of the presently preferred embodimentsthereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerProAspLysGlnMetAlaValLeuProArgArgGluArgAsnArg                              151015                                                                        GlnAlaAlaAlaAlaAsnProGluAsnSerArgGlyLysGlyArgArg                              202530                                                                        GlyGlnArgGlyLysAsnArgGlyCysValLeuThrAlaIleHisLeu                              354045                                                                        AsnValThrAspLeuGlyLeuGlyTyrGluThrLysGluGluLeuIle                              505560                                                                        PheArgTyrCysSerGlySerCysAspAlaAlaGluThrThrTyrAsp                              65707580                                                                      LysIleLeuLysAsnLeuSerArgAsnArgArgLeuValSerAspLys                              859095                                                                        ValGlyGlnAlaCysCysArgProIleAlaPheAspAspAspLeuSer                              100105110                                                                     PheLeuAspAspAsnLeuValTyrHisIleLeuArgLysHisSerAla                              115120125                                                                     LysArgCysGlyCysIle                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCT60                GCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGT120               TGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAG180               GAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGAC240               AAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCA300               TGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTAC360               CATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATC402                                 __________________________________________________________________________

What is claimed is:
 1. A method for treating injury or degeneration ofcells of the inner ear, comprising administering a glial cellline-derived neurotrophic factor (GDNF) protein product comprising anamino acid sequence set forth in SEQ ID NO:1, wherein said GDNF proteinproduct promotes the survival or function of cochlear hair cells andauditory neurons of the inner ear.
 2. The method of claim 1, whereinsaid auditory neurons are spiral ganglion neurons.
 3. The method ofclaim 1, wherein the GDNF protein product further comprises an aminoterminal methionine.
 4. The method of claim 1, wherein the GDNF proteinproduct is administered at a dose of about 1 μg/kg/day to about 100mg/kg/day.
 5. A method for treating injury or degeneration of cells ofthe inner ear comprising administering a glial cell line-derivedneurotrophic factor (GDNF) protein product comprising an amino acidsequence which is in excess of 70% identical to an amino acid sequenceset forth in SEQ ID NO:1 when up to four gaps in a length of 100 aminoacids may be introduced to assist in that alignment, wherein said GDNFprotein product promotes the survival or function of cochlear hair cellsand auditory neurons of the inner ear.
 6. The method of claim 5, whereinthe GDNF protein product further comprises an amino terminal methionine.7. The method of claim 5, wherein the GDNF protein product isadministered at a dose of about 1 μg/kg/day to about 100 mg/kg/day. 8.The method of claim 5, wherein said auditory neurons are spiral ganglionneurons.